The goal of the proposed research is to elucidate the mechanism by which antidiuretic hormone (ADH) promotes increased net water transport across the apical membranes of epithelial tissues. In toad urinary bladder at the mucosal border, ADH-induced exocytosis is associated with increasing permeability and ADH-induced endocytosis is associated with decreasing permeability. Our specific aim is to determine the role in water transport of membrane-delimited electron-dense granules and endocytic tubules in the affected G cells. ADH causes significant exocytosis at the initiation of the hydroosmotic response. Approximately 40 percent of the granules are released during a single stimulation adding apical glycocalyx and enough membrane to potentially double the surface. To evaluate the permeability characteristics of granules and its chemical basis, granules will be isolated free of other organelles. Granule osmotic behavior will be assessed using spectrophotometry and microscopy as part of our evaluation of heir physiological role. Using Ca electrodes and radioactive uptake we will evaluate the interactions between Ca2+ and granules. Protein and lipid contents of granule and plasma membrane fractions will be compared. Antibodies made tothe granules will be used in immunocytochemical identification of relationships between granules, endocytic tubules and plasma membranes of stimulated and unstimulated bladder. The bladder's cycle of exocytosis and endocytosis may conserve permeable membrane. Whether the G-cell conserves endocytic tubules and reuses them for multiple hormone responses will be examined by visualizing ADH-induced endocytic membrane retrieval in living bladder using fluorescent markers, Nomarski optics, video intensification and computerized image analysis. Osmotic gradients determine the rate of hydroosmosis in the bladder and the process of membrane fusion in other systems. In bladders subjected in vitro to transepithelial osmotic gradients the linkage of exocytosis, endocytosis and hydroosmosis will be assessed using electron microscopy, cytochemistry and physiology. This integrated physiological, biochemical, cytochemical and fine structural investigation into the cellular basis for conservation of water and ions by the toad urinary bladder will provide insight into corresponding mechanisms used in the inaccessible homologous mammalian distal nephron.